I have received many enquiries into the procedure we use for plasmid preps
(referred in an earlier posting entitled "Alas, poor magic! I knew thee well)
Below is the rapid boiling method (that is hardly new!) that we use. We have
found it to work very well for both sequencing and restriction analysis. But do
not use HB101 strain unless you incorporate a phenol extraction into the
procedure to inactivate the heat-resistant nuclease present in this strain.
Good luck!
Geoff Neale
Dept. of Virology and Molecular Biology Internet: neale at mbcf.stj.org
St. Jude Children's Research Hospital Phone: (901) 522-0400
Memphis, TN Fax: (901) 523-2622
Rapid boiling method for plasmid DNA
(1) Grow 2-3 ml of bacteria (see note (i) below) in 2 x LB or Superbroth + AMP
O/N in 50 ml tube.
(2) Transfer 1.5 ml to microfuge tube and spin 20-30 sec. Remove supernatant.
(3) Vortex cells to resuspend them in remaining supernatant. See note (ii)
below.
(4) Add 300 ul of triton buffer: 5% Triton X-100, 8% sucrose, 50 mM Tris pH
8.0, 50 mM EDTA. Vortex again to resuspend cells completely.
(5) Add 25 ul of 10 mg/ml lyzozyme. Vortex 2 sec.
(6) Boil 90-120 sec. You can use a heat block at 95 C.
(7) Spin 10 min.
(8) Transfer supernatant to new tube (see note (iii) below), and add 230 ul
(equal volume) of isopropanol. Mix, spin 5 min
(9) Wash pellet 2 x 500 ul of ethanol (without spinning again), and dry under
vacuum.
(10) Resuspend in 20 ul of TE or water. Use 2-4 ul for sequencing or
restriction
digest.
Notes:
(i) Do not use this procedure for HB101 cells. They contain a heat-stable
nuclease that will degrade your DNA with storage, or digestion. We have
obtained
very good results with high copy number plasmids (eg pBluescript, pGEM vectors)
in DH5 alpha and DH10B cells.
(ii) Be sure that the cell pellet is resuspended thoroughly before boiling.
Resuspending the cells in the remaining culture supernatant is the best way to
ensure complete resuspension.
(iii) After boiling the pellet should appear gooey and can be removed using a
toothpick leaving the supernatant behind, thus avoiding using another tube.
(iv) This procedure can be scaled up for 100 ml cultures:
Use 15 ml Triton solution in step 4
Add 2.5 ml of lysozyme in step 5
Use heat seal bag in boiling water bath in step 6
Spin 10,000 rpm for 15 min in Sorvall SS-34 (or equivalent) in step 7
Add 11.5 ml isopropanol in step 8
Wash with 25 ml ethanol step 9
Resuspend in 1 ml volume in step 10